Peptides mimicking hiv-1 viral epitopes in the v2 loop for the gp120 surface envelope glycoprotein

ABSTRACT

The present invention relates to an isolated immunogenic peptide comprising a V2 loop fragment from HIV surface envelope glycoprotein gp120. This peptide binds specifically with antibodies in blood of patients vaccinated with a vaccine that has shown protection from HIV-1 infection, does not react with blood of matched patients who did not receive the vaccine, and can, therefore, elicit anti-HIV-1 antibodies which protect against HIV-1 infection. Other aspects of the present invention relate to an isolated immunogenic polypeptide comprising the peptide inserted into an immunogenic scaffold protein, a vaccine composition comprised of the immunogenic peptide and an immunologically or pharmaceutically acceptable vehicle or excipient as well as methods of inducing an immune response against HIV-1 and methods of detecting HIV-1.

This application is a divisional application of U.S. patent applicationSer. No. 13/612,300, filed Sep. 12, 2012, and claims the benefit of U.S.Provisional Patent Application Ser. No. 61/533,424, filed Sep. 12, 2011,which is hereby incorporated by reference in its entirety.

The subject matter of this application was made with support from theUnited States National Institutes of Health Grant No. R01-A1084119. TheU.S. government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to a peptide mimicking HIV-1 viralepitopes in the V2 loop for the gp120 surface envelope glycoprotein.

BACKGROUND OF THE INVENTION

Human Immunodeficiency Virus-1 (HIV-1) infection has been reportedthroughout the world in both developed and developing countries. HIV-2infection is found predominately in West Africa, Portugal, and Brazil.At the end of 2008, an estimated 1,178,350 persons aged 13 and olderwere living with HIV infection in the United States. Of those, 20% hadundiagnosed HIV infections (CDC, “HIV Surveillance—United States,1981-2008,” MMWR 60(21); 689-693 (2008), which is hereby incorporated byreference in its entirety).

The HIV viruses are members of the Retroviridae family and, moreparticularly, are classified within the Lentivirinae subfamily. Likenearly all other viruses, the replication cycles of members of theRetroviridae family, commonly known as the retroviruses, includeattachment to specific cell receptors, entry into cells, synthesis ofproteins and nucleic acids, assembly of progeny virus particles(virions), and release of progeny viruses from the cells. A uniqueaspect of retrovirus replication is the conversion of thesingle-stranded RNA genome into a double-stranded DNA molecule that mustintegrate into the genome of the host cell prior to the synthesis ofviral proteins and nucleic acids.

HIV encodes a number of genes including three structural genes—gag, pol,and env—that are common to all retroviruses. The envelope protein ofHIV-1 is a glycoprotein of about 160 kd (gp160). During virus infectionof the host cell, gp160 is cleaved by host cell proteases to form gp120and the integral membrane protein, gp41. The gp41 portion is anchored inthe membrane bilayer of virion, while the gp120 segment protrudes intothe surrounding environment. gp120 and gp41 are more covalentlyassociated, and free gp120 can be released from the surface of virionsand infected cells. The gp120 polypeptide is also instrumental inmediating entry into the host cell.

Historically, viral vaccines have been enormously successful in theprevention of infection by a particular virus. Therefore, when HIV wasfirst isolated, there was a great amount of optimism that an HIV vaccinewould be developed quickly. However, this optimism quickly faded,because a number of unforeseen problems emerged.

It is widely thought that a successful vaccine should be able to inducea strong, broadly neutralizing antibody response against diverse HIV-1.Neutralizing antibodies, by attaching to the incoming virions, canreduce or even prevent their infectivity for target cells and preventthe cell-to-cell spread of virus in tissue. Conventional wisdom suggeststhat “constant” rather than “variable” regions of Env would induce themost broadly reactive antibodies. The failure of the Vaxgen HIV vaccinetrial demonstrated that the sequence-conserved regions of HIV gp120 donot induce protective neutralizing antibodies, since these regions werepresent in the gp120 immunogen used in that study. The failure of theSTEPS HIV vaccine demonstrated that cellular immunity utilizing non-Envdeterminants is not protective. Thus, one could conclude that targetingthe sequence-conserved (including non-Env and the core of Env) regionsof the HIV-1 virus for protective immunity will not work. Thus, thereremains a need for envelope antigens that can elicit an immunologicalresponse in a subject against multiple HIV strains and subtypes, forexample when administered as a vaccine.

The present invention is directed to overcoming deficiencies of priorapproaches to addressing HIV infection.

SUMMARY OF THE INVENTION

The present invention relates to an isolated immunogenic peptidecomprising a V2 loop fragment from HIV surface envelope glycoproteingp120. This peptide binds specifically with antibodies in blood ofpatients vaccinated with a vaccine that has shown protection from HIV-1infection, does not react with blood of matched patients who did notreceive the vaccine, and can, therefore, elicit anti-HIV-1 antibodieswhich protect against HIV-1 infection.

Other aspects of the present invention relate to an isolated immunogenicpolypeptide comprising the peptide inserted into an immunogenic scaffoldprotein, a vaccine composition comprised of the immunogenic peptide andan immunologically or pharmaceutically acceptable vehicle or excipientas well as methods of inducing an immune response against HIV-1 andmethods of detecting HIV-1.

The RV144 HIV-1 vaccine trial was the first to demonstrate evidence ofprotection against HIV-1 infection, with an estimated vaccine efficacyof 31.2% (Rerks-Ngarm et al., “Vaccination with ALVAC and AIDSVAX toPrevent HIV-1 Infection in Thailand,” N Engl J Med. 361:2209-2220(2009), which is hereby incorporated by reference in its entirety). Thisvaccine consisted of four doses of a recombinant canary pox primingimmunogen, ALVAC-HIV (vCP1521), and two doses of AIDSVAX® B/E,recombinant HIV-V gp120 proteins from HIV-1 subtype B and circulatingrecombinant form 01AE (CRF01_AE).

In order to identify correlates of risk of HIV-1 infection in RV144, twosequential sets of analyses of plasma specimens from study participantswere conducted (Haynes et al., “Immune Correlates Analysis of theALVAC-AIDSVAX HIV-1 Vaccine Efficacy Trial,” N Engl J Med. 366:1275-1286(2012), which is hereby incorporated by reference in its entirety). Thefirst was a series of pilot studies in which 32 types of immunologicassays were performed on sets of plasma and peripheral blood mononuclearcells from uninfected participants who had received either the placeboor the vaccine. Results from the pilot studies were used to selectassays for the subsequent case-control study of immune correlates ofinfection risk. Assays for the case-control study were chosen if theresults in the pilot studies showed low false positive rates, a broaddynamic range, low background reactivity, and low specimen volumerequirements (Haynes et al., “Immune Correlates Analysis of theALVAC-AIDSVAX HIV-1 Vaccine Efficacy Trial,” N Engl J Med. 366:1275-1286(2012), which is hereby incorporated by reference in its entirety).Seventeen assay types were selected for the case-control study, andthese generated results for 158 variables. To preserve maximalstatistical power, six were chosen as primary variables in thecase-control study and were analyzed by multivariate analysis. To expandthe search for immune correlates, all 158 variables were subsequentlyevaluated by univariate analyses.

Case-control specimens consisted of specimens drawn two weeks after thelast immunization from 41 infected vaccinees (cases) and from 205matched uninfected vaccinees (controls). Two of the six primaryvariables significantly correlated with HIV-1 infection risk in vaccinerecipients: 1) the level of plasma IgG antibodies reactive withgp70-V1V2, a scaffolded protein carrying the first and second variableregions of the HIV-1 gp120 envelope glycoprotein fused to murineleukemia virus gp70. Levels of antibodies specific for gp70-V1V2 werecorrelated inversely with the risk of infection; 2) the level of plasmaIgA antibodies reactive with a panel of 14 envelope glycoproteinscorrelated directly with risk of infection.

The participation of the V2 region of gp120 in the infectious process,and the role of V2 specific antibodies in protection from infection hasbeen the subject of investigation and controversy for nearly twodecades. Although, by definition, “variable” regions—like V2—vary inamino acid sequence, many residues in these regions do not vary, ortolerate only conservative changes (Zolla-Pazner et al,“Structure-Function Relationships of HIV-1 Envelope Sequence-VariableRegions Provide a Paradigm for Vaccine Design,” Nat Rev Immunol 10:527-535 (2010), which is hereby incorporated by reference in itsentirety). These conserved amino acids can form structural elements thatresult in immunologic cross-reactivity between diverse viruses; forexample many V2- and V3-specific antibodies are highly cross-reactivewith diverse HIV-1 envelopes (Gorny et al., “Repertoire of NeutralizingHuman Monoclonal Antibodies Specific For the V3 Domain of HIV-1 gp120,”J Immunol. 150: 635-643 (1993); Israel et al., “Prevalence of a V2Epitope in Clade B Primary Isolates and its Recognition by Sera fromHIV-1 Infected Individuals,” Aids 11: 128-130 (1997); Krachmarov et al.,“Antibodies That are Cross-Reactive for Human Immunodeficiency VirusType 1 Clade A and Clade B V3 Domains are Common in Patient Sera FromCameroon, but Their Neutralization Activity is Usually Restricted byEpitope Masking,” J Virol. 79: 780-790 (2005); Gorny et al., “Functionaland Immunochemical Cross-Reactivity of V2-Specific Monoclonal AntibodiesFrom Human Immunodeficiency Virus Type 1-Infected Individuals,” Virology427: 198-207 (2012); Nyambi et al., “Immunoreactivity of Intact Virionsof Human Immunodeficiency Virus Type 1 (HIV-1) Reveals the Existence ofFewer HIV-1 Immunotypes Than Genotypes,” J Virol 74: 10670-10680 (2000);Hioe et al., “Anti-V3 Monoclonal Antibodies Display Broad NeutralizingActivities Against Multiple HIV-1 Subtypes,” PLoS ONE 5: e10254 (2010),each of which is hereby incorporated by reference in its entirety).Moreover, the conserved structural features are required for theseregions to perform important biologic functions. Thus, for example,conserved elements within V2 participate in the formation of thebridging sheet (a constituent of the chemokine receptor binding site(Thali et al., “Characterization of Conserved HIV-type 1 gp120Neutralization Epitopes Exposed Upon gp120-CD4 Binding,” J Virol 67:3978-3988 (1993); Rizzuto et al., “A Conserved HIV gp120 GlycoproteinStructure Involved in Chemokine Receptor Binding,” Science 280:1949-1953 (1998); Kwong et al. “Structure of an HIV gp120 EnvelopeGlycoprotein in Complex With the CD4 Receptor and a Neutralizing HumanAntibody,” Nature 393: 648-659 (1998), each of which is herebyincorporated by reference in its entirety), and V2 contains a tripeptidemotif in the mid-loop region of V2 that is a putative α4β7 integrinbinding site (Arthos et al., “HIV-1 Envelope Protein Binds to andSignals Through lintegrin alpha4beta7, the Gut Mucosal Homing Receptorfor Peripheral T cells,” Nat Immunol 9: 301-309 (2008), which is herebyincorporated by reference in its entirety). Similarly, conservedelements of V3 contribute to its role in binding to the chemokinereceptor (Trkola et al., “CD4-Dependent, Antibody-Sensitive InteractionsBetween HIV-1 and its Co-Receptor CCR-5,” Nature 384: 184-187 (1996);Hill et al., “Envelope Glycoproteins From HIV-1, HIV-2 and SIV Can UseHuman CCR5 as a Coreceptor for Viral Entry and Make Direct CD4-DependentInteractions With This Chemokine Receptor,” J Virol 71: 6296-6304(1997), each of which is hereby incorporated by reference in itsentirety).

Antibodies specific for V2 occur in only ˜25-40% of HIV-infectedindividuals (Israel et al., “Prevalence of a V2 Epitope in Clade BPrimary Isolates and its Recognition by Sera from HIV-1 InfectedIndividuals,” Aids 11: 128-130 (1997); Kayman et al., “Presentation ofNative Epitopes in the V1/V2 and V3 Regions of Human ImmunodeficiencyVirus Type 1 gp120 by Fusion Glycoproteins Containing Isolated gp120Domains,” J Virol. 68: 400-410 (1994), each of which is herebyincorporated by reference in its entirety). Interestingly, thecross-reactivity of these antibodies does not require extensive mutationfrom the VH germ line since V2-specific monoclonal antibodies fromHIV-infected individuals display a mean 6.2% mutation frequency fromgerm line (Gorny et al., “Functional and Immunochemical Cross-Reactivityof V2-Specific Monoclonal Antibodies From Human Immunodeficiency VirusType 1-Infected Individuals,” Virology 427: 198-207 (2012), which ishereby incorporated by reference in its entirety) which is comparable toa mean 6.8% mutation frequency found in Abs from normal individuals(Tiller et al., “Autoreactivity in Human IgG+ Memory B Cells,” Immunity26: 205-213 (2007), which is hereby incorporated by reference in itsentirety). Thus, because a large body of data suggests that V2 may be asite of HIV-1 vulnerability, and because a strong antibody response togp70-V1V2 was correlated with reduced infection in the RV144 clinicalvaccine trial, a thorough analysis of all V2 antibody assays used in theRV144 immune correlates study was undertaken, as disclosed here.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the ELISA reactivity of human anti-V2 monoclonal antibodies697-D and 2158 with AIDSVAX subtype E and AIDSVAX subtype B gp120immunogens. The dashed line represents twice background levels.

FIG. 2 shows ELISA reactivity with gp70-V1V2 of plasma specimens used inthe pilot studies of the RV144 clinical vaccine trial (Set C). Theresults from one of three experiments are shown. The open and filledblue diamonds depict negative responses at weeks 0 and 26, respectively.The open and closed red circles depict positive responses at weeks 0 and26, respectively. Each vertical line connects a single patient'sspecimen drawn at Week 0 and Week 26. The specimens are ordered by thedifference in reactivity between the Week 0 and Week 26 specimens, withthe biggest increasers on the right. Plasma were tested at a finaldilution of 1:100, and a positive response was defined as being >0.276,the cut-off OD value which was defined as the mean+3 standard deviationsbased on values derived from vaccinees at week 0 (the pre-immunizationtime point).

FIG. 3 shows Spearman rank correlations between the V2 assays run in thecase-control study.

FIG. 4 shows ELISA reactivity of anti-V2 IgG antibodies in plasma fromvaccine recipients in the RV144 pilot study (Set L) against threedifferent V2 antigens (gp70-V1V2, V2 cyclic peptide from clade E strain92TH023, and the K178 V2 peptide) run in parallel in the same assay at aplasma dilution of 1:50. Data shown are from one of two experiments.Plasma from placebo recipients were negative. Statistical comparisonsbetween groups for positive responders were performed using Student'st-test.

FIGS. 5A-B show boxplots showing ELISA reactivity of V2 peptides withplasma (diluted 1:100) from 80 vaccinees' specimens from the pilot study(Set C). The distributions of the reactivities are shown where the leftedge of each box equals the 25th percentile; the vertical line in eachbox is the 50th percentile, and the right edge of each box equals the75th percentile. The boxplots were prepared using the scientificgraphing program, GraphPad Prism, with “whiskers” showing the minimumand maximum responses. FIG. 5A shows four 21-mer N-terminal biotinylatedpeptides (Peptides 1-4 (SEQ ID NOS: 3 to 6)) were selected on the basisof a bioinformatics analysis of V2 sequences from the LANL HIV Database.FIG. 5B shows a second peptide panel designed upon inspection of theamino acids in Peptides 1-4 (SEQ ID NOS: 3 to 6) in FIG. 5A revealedamino acids that distinguish Peptides 1 (SEQ ID NO: 3) and 3 (SEQ ID NO:5) from 2 (SEQ ID NO: 4) and 4 (SEQ ID NO: 6); these appear at positions165, 169, 172, and 174. To maximally enhance the availability of theepitopes on the peptides used in the fine mapping of the V2 antibodies,a spacer of three glycines was inserted between the biotin tag at theN-terminus of the peptide and the V2 sequences.

FIG. 6 shows a ribbon diagram of the backbone fold of the V1V2 domainbound to the CDR H3 loop of monoclonal antibody PG9 (transparentstippled spheres are the atoms of the PG9 CDR H3). The ribbon backbonesof amino acids 165-176 are identical to Peptide 6 (SEQ ID NO: 7), islabeled C. The strands are labeled A-D according to the conventionestablished recently (McLellan et al., “Structure of HIV-1 gp120 V1/V2Domain With Broadly Neutralizing Antibody PG9,” Nature 480:336-343(2011), which is hereby incorporated by reference in its entirety).

FIG. 7 shows estimated odds ratios (ORs) and 95% confidence intervalsfor each of the V2 assays. Data are derived from the categoricalanalyses shown in Table 2. Estimated ORs compare subgroups defined byhigh vs. low responses except for comparisons for analyses of IgA V2A244 K178 and V2 MN which compare positive vs. negative responses. Forevaluation of biotinylated V2 peptide 6 (SEQ ID NO: 7), comparison isbetween high and negative responses.

FIGS. 8A-B show microarray analysis of the V2 antibody response inplasma from vaccinees in the case-control study. FIG. 8A shows anaggregate response across all sub-types averaged across vaccinees as afunction of HxB2 positions. An individual aggregate response is computedusing a sliding window mean statistic of 9 amino acids, i.e., peptideswith HxB2 positions of 9 contiguous amino acids averaged together. InFIG. 8B, the actual sequence of each of the overlapping 15-mers (SEQ IDNOS: 17 to 65) spanning V2 positions 160-183 is shown. The seven sets ofpeptides represent the consensus V2 regions of HIV-1 Group M and ofsubtypes A, B, C, D, CRF01_AE and CRF02_AG. Peptides are shadedaccording to their average response across all vaccinees, with a scaleof 0 (white) to 1.8 (black). The V2 sequence of HxB2 is shown above thegraph with the corresponding HxB2 numbering, and this numbering is alsoshown on the x-axis. The bold arrow indicates that the estimatedaggregate response is highest when centered at position 173, thoughpeptides centered on position 170, i.e., with the V2 peptide spanningresidues 163-177 have the highest response. All responses are calculatedusing normalized intensities and by subtracting the intensities ofbaseline pre-bleeds.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an isolated immunogenic peptidecomprising a V2 loop fragment from HIV surface envelope glycoproteingp120. This peptide binds specifically with antibodies in blood ofpatients vaccinated with a vaccine that has shown protection from HIV-1infection, does not react with blood of matched patients who did notreceive the vaccine, and can, therefore, elicit anti-HIV-1 antibodieswhich protect against HIV-1 infection.

In accordance with this aspect of the present invention, suitableisolated immunogenic peptides include peptides of the amino acidsequence X₁X₂DX₃X₄X₅X₆X₇YX₈X₉X₁₀X₁₁X₁₂ (SEQ ID NO: 1), where X₁ is L, V,I, M, F, W or A; X₂ is any amino acid; X₃ is R, K or H; X₄ is K, D, E,R, H, S, T, C, N, Q, Y, A, V or M; X₅ is K, D, E, R, H, S, T, C, N, Q, Yor A; X₆ is K, D, E, R, H, S, T, C, N, Q, Y or A; X₇ is L, V, I, M, F,W, A or E; X₈ is K, D, E, R, H, S, T, C, N, Q, Y or A; X₉ is L, V, I, M,F, W or A; X₁₀ is F or T, X₁₁ is K, D, E, R, H, S, T, C, N, Q, Y or A;X₁₂ is K, D, E, R, H, S, T, C, N, Q, Y or A. Examples of specificimmunogenic peptides in accordance with the present invention includethe following amino acid sequences: LRDKKQRVYSLFYK (SEQ ID NO: 7),IRDKKQRVYSLFYK (SEQ ID NO: 11), LRDKVQRVYSLFYK (SEQ ID NO: 12),LRDKKQREYSLFYK (SEQ ID NO: 13), LRDKKQRVYALFYK (SEQ ID NO: 14), orLQNKKQQVYSLFYQ (SEQ ID NO: 15).

Another aspect of the present invention is an isolated immunogenicpeptide including the amino acid sequence of SEQ ID NO: 2. In accordancewith this aspect of the present invention, suitable isolated immunogenicpeptides include peptides of the amino acid sequence of SEQ ID NO: 1with X₁₁ as any amino acid other than Y. In particular, SEQ ID NO: 2 hasthe following amino acid sequence: X₁X₂DX₃X₄X₅X₆X₇YX₈X₉X₁₀X₁₁X₁₂, whereX₁ is L, V, I, M, F, W or A; X₂ is any amino acid; X₃ is R, K or H; X₄is K, D, E, R, H, S, T, C, N, Q, Y, A, V or M; X₅ is K, D, E, R, H, S,T, C, N, Q, Y or A; X₆ is K, D, E, R, H, S, T, C, N, Q, Y or A; X₇ is L,V, I, M, F, W, A or E; X₈ is K, D, E, R, H, S, T, C, N, Q, Y or A; X₉ isL, V, I, M, F, W or A; X₁₀ is F or T, X₁₁ is any amino acid other thanY; X₁₂ is K, D, E, R, H, S, T, C, N, Q, Y or A.

In another aspect of the present invention, the isolated immunogenicpeptide comprises the amino acid sequence of LRDKMQKVYALTYK (SEQ ID NO:16). This is a sequence that does not occur in nature and has amutation, relative to the amino acid sequence of SEQ ID: 1, whichrequires Y at position X₁₁. This destroys a cathepsin protease cleavagesite.

The present invention also relates to an isolated immunogenicpolypeptide of the present invention comprising the isolated immunogenicpeptide described above and an immunogenic scaffold protein. Thepolypeptide has a conformation that is recognized by, and bound by, abroadly neutralizing anti-HIV-1 antibody.

As used herein, a “broadly neutralizing” antibody or antibody responseis an antibody or response that results in binding and neutralization ofat least one group of heterologous HIV-1 viruses that are members of adifferent subtype or clade than that of the source of the immunizingantigen. The scaffold protein can be one that is highly immunogenic andcapable of enhancing the immunogenicity of any heterologous sequencesfused to/inserted in it. Suitable scaffold proteins include, withoutlimitation, a cholera toxin and an enterotoxin.

In one embodiment of the present invention, the scaffold protein ischolera toxin subunit B (CTB). CTB is highly immunogenic and has beenused in fusion constructs to enhance immunogenicity of its fusionpartner polypeptide or peptide (McKenzie et al., “Cholera Toxin BSubunit as a Carrier to Stimulate a Mucosal Immune Response,” J Immunol.133:1818-1824 (1984); Czerkinsky et al., “Oral Administration of aStreptococcal Antigen Coupled to Cholera Toxin B Subunit Evokes StrongAntibody Responses in Salivary Glands and Extramucosal Tissues,” InfectImmun. 57:1072-1077 (1989), each of which is hereby incorporated byreference in its entirety). CTB has also been described as a mucosaladjuvant for vaccines (Areas et al., “Expression and Characterization ofCholera Toxin B-Pneumococcal Surface Adhesin A Fusion Protein inEscherichia Coli: Ability of CTB-PsaA to Induce Humoral Immune Responsein Mice,” Biochem Biophys Res Commun. 321:192-196 (2004), which ishereby incorporated by reference in its entirety).

In another embodiment of the present invention, the scaffold protein isan E. coli enterotoxin, preferably heat labile entertoxin. This proteinis also highly immunogenic and has been used in fusion constructs toenhance immunogenicity of its fusion partner polypeptide or peptide(Lipscombe et al., “Intranasal Immunization Using the B Subunit of theEscherichia Coli Heat Labile Toxin Fused to an Epitope of the BordetellaPertussis P.69 Antigen,” Mol Microbiol. 5:1385-1392 (1991), which ishereby incorporated by reference in its entirety).

An important factor for the immunogenic property of CTB and heat labileenterotoxin is their binding to GM1 ganglioside, which is present on thesurface of mucosal cells. This results in its propensity to inducemucosal immunity and is highly desirable for an HIV immunogen orvaccine, because infection commonly occurs via a mucosal route. Inaddition, the availability of structural information of these proteinsallows protein design that avoids or minimizes disruption of the GM1binding site, thereby preserving the inherent immunogenicity of thesepolypeptides.

In accordance with this aspect of the present invention, the immunogenicpeptide can be inserted directly into the scaffold's tertiary structure.This yields a polypeptide in which an exceptionally high fraction of themolecular surface presents V2 epitopes that are recognized bybroadly-reactive neutralizing anti-gp120 antibodies and can elicitanti-HIV-1 antibody responses that preferably are broadly-reactive andneutralize the virus. Molecular modeling is used to test in-silico,whether various insertion positions in the scaffold and different looplengths result in loop conformations that present the epitopes.Specifically, there are two approaches. Firstly, the scaffold is scannedfor amino-acid positions that can be superimposed on the termini of theloop as observed in the V2/antibody complex. When superposition withinsmall tolerances (<0.5 .ANG. root mean square deviation (RMSD) for theterminal residues is achieved, the model is evaluated for the absence ofclashes with the scaffold structure. Secondly, the loop is inserted in arandom conformation and subjected to conformational sampling. Low energyconformations generated during sampling are compared to the desired V2conformation as observed in the V2/antibody complex. Sampling is over arestricted energy range. When the construct is such that conformationswithin 1.0 .ANG. backbone RMSD of the desired V2 conformation areidentified in the simulation, a model of the immunogen-antibody complexis built to ensure that the scaffold does not interfere with the V2loop/antibody binding.

The isolated immunogenic peptides described above may also exist in acyclized form. These cyclic peptides can be synthesized and include twocysteine residues that bond via a disulfide linkage forming the cyclicpeptide. Alternatively, the peptide may be cyclized by chemical meanswithout relying upon disulfide bonding of two cysteine residues, forexample, by introduction of a linker.

The cyclic peptide compositions of the present invention may besynthesized using ordinary skill in the art of organic synthesis andpeptide synthesis. Methods for restricting the secondary structure ofpeptides and proteins are highly desirable for the rational design oftherapeutically useful conformationally-restricted (or “locked”)pharmacophores. The purely chemical approaches for restricting secondarystructure often require extensive multistep syntheses (Olson, G. L., J.Am. Chem. Soc. 112:323 (1990)). An alternative approach involvesinstalling covalent bridges in peptides. However, due to the sensitivityof the peptide backbone and side chains, this method necessitatescareful protection/deprotection strategies.

The general guiding principles determining the design of useful cyclicpeptides are well-known in the art and are dictated by the need tomaintain the antibody reactivity and immunogenicity of the V2 peptide,particularly for induction of broadly reactive, neutralizing antibodieswhile enhancing its stability as well as the ability to be inserted intoa desired scaffold protein without disrupting the “function” of thelatter, i.e., immunogenicity and other binding characteristics of thescaffold such as the binding of recombinant V2-CTB to the glycolipidtargets of CTB. In addition to testing a cyclic peptide serologically,it may be analyzed more extensively by structural (biophysical)techniques, such as NMR spectroscopy or X-ray crystallographic methods,in solution or when bound to a characterizing broadly-reactiveneutralizing monoclonal antibody such as 447-52D (see publicationWO04/069863, which is hereby incorporated by reference in its entirety)

The one or more peptides in the present invention can be synthesized bysolid phase or solution phase peptide synthesis, recombinant expression,or can be obtained from natural sources. Automatic peptide synthesizersare commercially available from numerous suppliers, such as AppliedBiosystems, Foster City, Calif. Standard techniques of chemical peptidesynthesis are well known in the art (see e.g., SYNTHETIC PEPTIDES: AUSERS GUIDE 93-210 (Gregory A. Grant ed., 1992), which is herebyincorporated by reference in its entirety). Peptide production viarecombinant expression can be carried out using bacteria, such as E.coli, yeast, insect cells or mammalian cells and expression systems.Procedures for recombinant protein/peptide expression are well known inthe art and are described by Sambrook et al., Molecular Cloning: ALaboratory Manual (C.S.H.P. Press, NY 2d ed., 1989), which is herebyincorporated by reference in its entirety.

Recombinantly expressed peptides can be purified using any one ofseveral methods readily known in the art, including ion exchangechromatography, hydrophobic interaction chromatography, affinitychromatography, gel filtration, and reverse phase chromatography. Thepeptide is preferably produced in purified form (preferably at leastabout 80% to 85% pure, more preferably at least about 90% or 95% pure)by conventional techniques. Depending on whether the recombinant hostcell is made to secrete the peptide into growth medium (see U.S. Pat.No. 6,596,509 to Bauer et al., which is hereby incorporated by referencein its entirety), the peptide can be isolated and purified bycentrifugation (to separate cellular components from supernatantcontaining the secreted peptide) followed by sequential ammonium sulfateprecipitation of the supernatant. The fraction containing the peptide issubjected to gel filtration in an appropriately sized dextran orpolyacrylamide column to separate the peptides from other proteins. Ifnecessary, the peptide fraction may be further purified by HPLC.

Another aspect of the present invention is directed to an immunogenicvaccine composition comprising the linear or cyclized isolatedimmunogenic peptides or polypeptides described above, and animmunologically and pharmaceutically acceptable vehicle or excipient.

Suitable vehicles and excipients are described in REMINGTON'SPHARMACEUTICAL SCIENCE (19th ed., 1995), which is hereby incorporated byreference in its entirety. The incorporation of such immunologically andpharmaceutically acceptable components depends on the intended mode ofadministration and therapeutic application of the pharmaceuticalcomposition. Typically, however, the vaccine composition will include apharmaceutically-acceptable, non-toxic carrier or diluent, which aredefined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the composition.Exemplary carriers or diluents include distilled water, physiologicalphosphate-buffered saline, Ringer's solutions, dextrose solution, andHank's solution.

Vaccine compositions can also include large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such as latexfunctionalized sepharose, agarose, cellulose), polymeric amino acids,amino acid copolymers, and lipid aggregates (such as oil droplets orliposomes).

The vaccine composition of the present invention may also besupplemented with an immunostimulatory cytokine. Preferred cytokines areGM-CSF (granulocyte-macrophage colony stimulating factor), interleukin1, interleukin 2, interleukin 12, interleukin 18, or interferon-gamma.

The vaccine composition of the present invention can further contain anadjuvant. One class of preferred adjuvants is aluminum salts, such asaluminum hydroxide, aluminum phosphate, or aluminum sulfate. Suchadjuvants can be used with or without other specific immunostimulatingagents such as MPL or 3-DMP, QS-21, flagellin, polymeric or monomericamino acids such as polyglutamic acid or polylysine, or pluronicpolyols. Oil-in-water emulsion formulations are also suitable adjuvantsthat can be used with or without other specific immunostimulating agentssuch as muramyl peptides (e.g.,N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE),N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) Theramide®, or other bacterial cell wallcomponents). A suitable oil-in-water emulsion is MF59® (containing 5%Squalene, 0.5% Tween® 80, and 0.5% Span® 85 (optionally containingvarious amounts of MTP-PE) formulated into submicron particles using amicrofluidizer such as Model 110Y Microfluidizer® (Microfluidics, NewtonMass.) as described in WO90/14837 to Van Nest et al., which is herebyincorporated by reference in its entirety. Other suitable oil-in-wateremulsions include SAF (containing 10% Squalene, 0.4% Tween® 80, 5%Pluronic®-blocked polymer L121, and thr-MDP, either microfluidized intoa submicron emulsion or vortexed to generate a larger particle sizeemulsion) and Ribiadjuvant system (RAS; containing 2% squalene, 0.2%Tween® 80, and one or more bacterial cell wall components). Anotherclass of suitable adjuvants are saponin adjuvants, such as Stimulon®(QS-21) or particles generated therefrom such as ISCOMs(immunostimulating complexes) and ISCOMATRIX™. Other suitable adjuvantsinclude incomplete or complete Freund's Adjuvant (IFA). Such adjuvantsare generally available from commercial sources.

Another aspect of the present invention relates to a method of inducinga broadly neutralizing antibody response against a V2 epitope of HIV-1gp120 in a subject. This method comprises administering to the subjectthe immunogenic peptide, cyclized peptide, or polypeptides, describedabove, under conditions effective to induce, in the subject, aneutralizing antibody response against the V2 epitope of the HIV-1gp120. In a preferred embodiment of this aspect, the selected subject isHIV-1 positive.

In accordance with this aspect of the present invention, a neutralizingantibody response is an antibody or response that results in binding andneutralization of at least one group of heterologous HIV-1 viruses thatare members of a different subtype or clade than that of the source ofthe immunizing antigen. Such a response is an active response induced byadministration of the immunogenic peptide or polypeptide and representsa means for vaccination against HIV-1.

An MT-2 assay can be performed to measure neutralizing antibodyresponses. Antibody-mediated neutralization of selected strains orisolates of HIV-1 can be measured in an MT-2 cell-killing assay(Montefiori et al., “Evaluation of Antiviral Drugs and NeutralizingAntibodies to Human Immunodeficiency Virus By a Rapid and SensitiveMicrotiter Infection Assay,” J Clin Microbiol. 26(2):2310-235 (1988),which is hereby incorporated by reference in its entirety).HIV-1.sub.IIIB and HIV-1.sub.MN induce the formation of syncytia in MT-2cells. The inhibition of the formation of syncytia by the sera shows theactivity of neutralizing antibodies present within the sera, induced byvaccination. Immunized test and control sera can be exposed tovirus-infected cells (e.g. cells of the MT-2 cell line). Neutralizationcan be measured by any method that determines viable cells, such asstaining, e.g., with Finter's neutral red. Percentage protection can bedetermined by calculating the difference in absorption between testwells (cells+virus) and dividing this result by the difference inabsorption between cell control wells (cells only) and virus controlwells (virus only). Neutralizing titers may be expressed, for example,as the reciprocal of the plasma dilution required to protect at least50% of cells from virus-induced killing.

Another aspect of the present invention relates to a method of inducinga protective and non-neutralizing antibody response against a V2 epitopeof HIV-1 gp120 in a subject. This method comprises administering to thesubject the immunogenic peptide, cyclized peptide, or polypeptides, asdescribed above, under conditions effective to induce, in the subject, aprotective, non-neutralizing antibody response against the V2 epitope ofthe HIV-1 gp120. In a preferred embodiment of this aspect, the selectedsubject is HIV-1 positive.

In accordance with this aspect of the present invention,non-neutralizing antibodies will not impair virus entry into cells.However, a non-neutralizing antibody response will triggerantibody-dependent cell-mediated viral inhibition (ADCVI), which may beeffective against HIV-1 (Asmal et al., “Antibody Dependent Cell MediatedViral Inhibition Emerges After Simian Immunodeficiency Virus SIVmac251Infection of Rhesus Monkeys Coincident With gp140-Binding Antibodies andis Effective Against Neutralization Resistant Viruses,” J Virol. 85(11)5465-5475 (2011), which is hereby incorporated by reference in itsentirety).

ADCVI can be measured by infecting polybrene-treated CEM.NKR-CCR5 cells(National Institutes of Health AIDS Research and Reference Program) witha clinical strain of HIV-1 (HIV-1_(92US657)). The addition of effectorcells from healthy donors and sera from vaccine subjects to the infectedtarget cells can lead to ADCVI. Levels of p24, as determined by ELISAcan determine the presence of ADCVI (Forthal et al., “Recombinant gp120Vaccine-Induced Antibodies Inhibit Clinical Strains of HIV-1 in thePresence of Fc Receptor Bearing Effector Cells and Correlate Inverselywith HIV Infection Rate,” J of Immunol. 178(10): 6596-6603 (2007), whichis hereby incorporated by reference in its entirety).

Another aspect of the present invention relates to a method of inducingprotective antibodies against a V2 epitope of HIV-1 gp120 in a subject.This method comprises administering to the subject the immunogenicpeptide, cyclized peptide, or polypeptide, as described above, underconditions effective to induce, in the subject, a protective antibodyresponse against the V2 epitope of the HIV-1 gp120. In one embodiment,the selected subject is HIV-1 positive.

The presence of a protective humoral immunological response can bedetermined and monitored by testing a biological sample (e.g., blood,plasma, serum, urine, saliva, feces, CSF or lymph fluid) from thesubject for the presence of antibodies directed to the immunogeniccomponent of the administered pharmaceutical composition.

The immunization protocol preferably includes at least one priming dose,followed by one or multiple boosting doses administered over time. Anexemplary range for an immunogenically effective amount of the presentimmunogenic polypeptides is about 5 to about 500 μg/kg body weight. Apreferred range is about 10-100 μg/kg.

Compositions of the present invention can be administered by parenteral,topical, intravenous, oral, subcutaneous, intraperitoneal, intranasal,or intramuscular means. The most typical route of administration forcompositions formulated to induce an immune response is subcutaneous,although others can be equally effective. The next most common isintramuscular injection. This type of injection is most typicallyperformed in the arm or leg muscles. Intravenous injections as well asintraperitoneal injections, intra-arterial, intracranial, or intradermalinjections are also effective in generating an immune response.

The compositions of the present invention may be formulated forparenteral administration. Solutions or suspensions of the agent can beprepared in water suitably mixed with a surfactant such ashydroxypropylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof in oils. Illustrativeoils are those of petroleum, animal, vegetable, or synthetic origin, forexample, peanut oil, soybean oil, or mineral oil. In general, water,saline, aqueous dextrose and related sugar solution, and glycols, suchas propylene glycol or polyethylene glycol, are preferred liquidcarriers, particularly for injectable solutions. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms.

Vaccine formulations suitable for injectable use include sterile aqueoussolutions or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersions. In allcases, the form must be sterile and must be fluid to the extent thateasy syringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol), suitable mixtures thereof, and vegetable oils.

When it is desirable to deliver the vaccine of the present inventionsystemically, it may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions, or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing, and/or dispersing agents.

Intraperitoneal or intrathecal administration of the agents of thepresent invention can also be achieved using infusion pump devices suchas those described by Medtronic, Northridge, Calif. Such devices allowcontinuous infusion of desired compounds avoiding multiple injectionsand multiple manipulations.

In addition to the formulations described previously, the agents mayalso be formulated as a depot preparation. Such long acting formulationsmay be formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

The present invention is further directed to an isolated antibody raisedagainst the immunogenic peptide or polypeptide of the present invention.The isolated antibody of the present invention encompasses anyimmunoglobulin molecule that specifically binds the V2 epitope of HIV-1gp120. As used herein, the term “antibody” is meant to include intactimmunoglobulins derived from natural sources or from recombinantsources, as well as immunoreactive portions (i.e., antigen bindingportions) of intact immunoglobulins. The antibodies of the presentinvention may exist in a variety of forms including, for example,polyclonal antibodies, monoclonal antibodies, intracellular antibodies(“intrabodies”), antibody fragments (e.g. Fv, Fab and F(ab)2), as wellas single chain antibodies (scFv), chimeric antibodies and humanizedantibodies (Ed Harlow and David Lane, USING ANTIBODIES: A LABORATORYMANUAL (Cold Spring Harbor Laboratory Press, 1999); Houston et al.,“Protein Engineering of Antibody Binding Sites: Recovery of SpecificActivity in an Anti-Digoxin Single-Chain Fv Analogue Produced inEscherichia coli,” Proc Natl Acad Sci USA 85:5879-5883 (1988); Bird etal, “Single-Chain Antigen-Binding Proteins,” Science 242:423-426 (1988),each of which is hereby incorporated by reference in its entirety).

Antibodies of the present invention may also be synthetic antibodies. Asynthetic antibody is an antibody which is generated using recombinantDNA technology, such as, for example, an antibody expressed by abacteriophage. Alternatively, the synthetic antibody is generated by thesynthesis of a DNA molecule encoding and expressing the antibody of thepresent invention or the synthesis of an amino acid specifying theantibody, where the DNA or amino acid sequence has been obtained usingsynthetic DNA or amino acid sequence technology which is available andwell known in the art.

Methods for monoclonal antibody production may be carried out using thetechniques described herein or other well-known in the art (MONOCLONALANTIBODIES—PRODUCTION, ENGINEERING AND CLINICAL APPLICATIONS (Mary A.Ritter and Heather M. Ladyman eds., 1995), which is hereby incorporatedby reference in its entirety). Generally, the process involves obtainingimmune cells (lymphocytes) from the spleen of a mammal which has beenpreviously immunized with the antigen of interest (i.e., a polymerizedfirst or second peptide or fusion peptide) either in vivo or in vitro.

The antibody-secreting lymphocytes are then fused with myeloma cells ortransformed cells, which are capable of replicating indefinitely in cellculture, thereby producing an immortal, immunoglobulin-secreting cellline. Fusion with mammalian myeloma cells or other fusion partnerscapable of replicating indefinitely in cell culture is achieved bystandard and well-known techniques, for example, by using polyethyleneglycol (PEG) or other fusing agents (Milstein and Kohler, “Derivation ofSpecific Antibody-Producing Tissue Culture and Tumor Lines by CellFusion,” Eur J Immunol 6:511 (1976), which is hereby incorporated byreference in its entirety). The immortal cell line, which is preferablymurine, but may also be derived from cells of other mammalian species,is selected to be deficient in enzymes necessary for the utilization ofcertain nutrients, to be capable of rapid growth, and have good fusioncapability. The resulting fused cells, or hybridomas, are cultured, andthe resulting colonies screened for the production of the desiredmonoclonal antibodies. Colonies producing such antibodies are cloned,and grown either in vivo or in vitro to produce large quantities ofantibody.

Alternatively monoclonal antibodies can be made using recombinant DNAmethods as described in U.S. Pat. No. 4,816,567 to Cabilly et al, whichis hereby incorporated by reference in its entirety. The polynucleotidesencoding a monoclonal antibody are isolated from mature B-cells orhybridoma cells, for example, by RT-PCR using oligonucleotide primersthat specifically amplify the genes encoding the heavy and light chainsof the antibody. The isolated polynucleotides encoding the heavy andlight chains are then cloned into suitable expression vectors, whichwhen transfected into host cells such as E. coli cells, simian COScells, Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, monoclonal antibodies aregenerated by the host cells. Also, recombinant monoclonal antibodies orfragments thereof of the desired species can be isolated from phagedisplay libraries (McCafferty et al., “Phage Antibodies: FilamentousPhage Displaying Antibody Variable Domains,” Nature 348:552-554 (1990);Clackson et al., “Making Antibody Fragments using Phage DisplayLibraries,” Nature 352:624-628 (1991); and Marks et al., “By-PassingImmunization. Human Antibodies from V-Gene Libraries Displayed onPhage,” J. Mol. Biol. 222:581-597 (1991), which are hereby incorporatedby reference in their entirety).

The polynucleotide(s) encoding a monoclonal antibody can further bemodified using recombinant DNA technology to generate alternativeantibodies. For example, the constant domains of the light and heavychains of a mouse monoclonal antibody can be substituted for thoseregions of a human antibody to generate a chimeric antibody.Alternatively, the constant domains of the light and heavy chains of amouse monoclonal antibody can be substituted for a non-immunoglobulinpolypeptide to generate a fusion antibody. In other embodiments, theconstant regions are truncated or removed to generate the desiredantibody fragment of a monoclonal antibody. Furthermore, site-directedor high-density mutagenesis of the variable region can be used tooptimize specificity and affinity of a monoclonal antibody.

The monoclonal antibody of the present invention can be a humanizedantibody. Humanized antibodies are antibodies that contain minimalsequences from non-human (e.g., murine) antibodies within the variableregions. Such antibodies are used therapeutically to reduce antigenicityand human anti-mouse antibody responses when administered to a humansubject. In practice, humanized antibodies are typically humanantibodies with minimal to no non-human sequences. A human antibody isan antibody produced by a human or an antibody having an amino acidsequence corresponding to an antibody produced by a human.

Procedures for raising polyclonal antibodies are also well known.Typically, such antibodies can be raised by administering the peptide orpolypeptide containing the epitope of interest (i.e. polymerized firstor second peptides or fusion peptides) subcutaneously to rabbits (e.g.New Zealand white rabbits), goats, sheep, swine or donkeys which havebeen bled to obtain pre-immune serum. The antigens can be injected incombination with an adjuvant. The rabbits are bled approximately everytwo weeks after the first injection and periodically boosted with thesame antigen three times every six weeks. Polyclonal antibodies arerecovered from the serum by affinity chromatography using thecorresponding antigen to capture the antibody. This and other proceduresfor raising polyclonal antibodies are disclosed in Ed Harlow and DavidLane, USING ANTIBODIES: A LABORATORY MANUAL (Cold Spring HarborLaboratory Press, 1988), which is hereby incorporated by reference inits entirety.

In addition to whole antibodies, the present invention encompassesbinding portions of such antibodies. Such binding portions include themonovalent Fab fragments, Fv fragments (e.g., single-chain antibody,scFv), and single variable V_(H) and V_(L) domains, and the bivalentF(ab′)₂ fragments, Bis-scFv, diabodies, triabodies, minibodies, etc.These antibody fragments can be made by conventional procedures, such asproteolytic fragmentation procedures, as described in James Goding,MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE 98-118 (Academic Press,1983) and Ed Harlow and David Lane, ANTIBODIES: A LABORATORY MANUAL(Cold Spring Harbor Laboratory, 1988), which are hereby incorporated byreference in their entirety, or other methods known in the art.

It may further be desirable, especially in the case of antibodyfragments, to modify the antibody in order to increase its serumhalf-life. This can be achieved, for example, by incorporation of asalvage receptor binding epitope into the antibody fragment by mutationof the appropriate region in the antibody fragment or by incorporatingthe epitope into a peptide tag that is then fused to the antibodyfragment at either end or in the middle (e.g., by DNA or peptidesynthesis).

Antibody mimics are also suitable for use in accordance with the presentinvention. A number of antibody mimics are known in the art including,without limitation, those known as monobodies, which are derived fromthe tenth human fibronectin type III domain (10Fn3) (Koide et al., “TheFibronectin Type III Domain as a Scaffold for Novel Binding Proteins,” JMol Biol 284:1141-1151 (1998); Koide et al., “Probing ProteinConformational Changes in Living Cells by Using Designer BindingProteins: Application to the Estrogen Receptor,” Proc Natl Acad Sci USA99:1253-1258 (2002), each of which is hereby incorporated by referencein its entirety); and those known as affibodies, which are derived fromthe stable alpha-helical bacterial receptor domain Z of staphylococcalprotein A (Nord et al., “Binding Proteins Selected from CombinatorialLibraries of an alpha-helical Bacterial Receptor Domain,” NatureBiotechnol 15(8):772-777 (1997), which is hereby incorporated byreference in its entirety).

The present invention is further directed to a method of detectingwhether a subject is infected with HIV-1. This method includes providinga sample from the subject. The sample is contacted with the immunogenicpeptide described above under conditions effective to cause animmunogenic reaction between antibodies in the sample and theimmunogenic peptide. Any subject, where the contacting results in theimmunogenic reaction, is identified as being infected with HIV-1. Thediagnosis of HIV-1 is based on the detection of V2-specific antibodiesin the subject. The presence of antibodies reactive with the V2-specificpeptides can be detected using standard electrophoretic andimmunodiagnostic techniques, including immunoassays such as competition,direct reaction, or sandwich type assays. Such assays include, but arenot limited to: western blots; agglutination tests; enzyme-labeled andmediated immunoassays, such as ELISAs; biotin/avidin type assays;radioimmunoassays; immunoelectrophoresis; immunoprecipitation, etc. Thereactions generally involve using labels such as fluorescent,chemiluminescent, radioactive, or enzymatic labels or dye molecules, orother methods for detecting the formation of a complex between theantigen and the antibody.

EXAMPLES

The following examples are provided to illustrate embodiments of thepresent invention but they are by no means intended to limit its scope.

Materials and Methods for Examples 1-4

Specimens.

The initial pilot studies were performed using various sets of plasmafrom the RV144 participants which were selected randomly, evenlybalanced for men and women, and derived from participants at visit 1(pre-bleed), visit 8 (week 26 after the first immunization [two weeksafter the last immunization]), and visit 9 (52 weeks). The pilot studieswere performed with plasma sets C (SZP, PB), A and L (GT, BH), and Z(MR, NK). Subsequently, case-control plasma specimens, described above,were tested for the primary and secondary variables selected asdescribed above and in Haynes et al., “Immune Correlates Analysis of theALVAC-AIDSVAX HIV-1 Vaccine Efficacy Trial,” N Engl J Med. 366:1275-1286(2012), which is hereby incorporated by reference in its entirety.

Written informed consent and counseling was conducted as describedpreviously (Rerks-Ngarm et al., “Vaccination with ALVAC and AIDSVAX toPrevent HIV-1 Infection in Thailand,” N Engl J Med. 361:2209-2220(2009); Haynes et al., “Immune Correlates Analysis of the ALVAC-AIDSVAXHIV-1 Vaccine Efficacy Trial,” N Engl J Med. 366:1275-1286 (2012), eachof which is hereby incorporated by reference in its entirety), and theprotocol was reviewed by the ethics committees of the Thai Ministry ofPublic Health, the Royal Thai Army, Mahidol University, and the HumanSubjects Research Review Board of the U.S. Army Medical Research andMateriel Command. It was also independently reviewed and endorsed by theWorld Health Organization and the Joint United Nations Program onHIV/AIDS and by the AIDS Vaccine Research Working Group of the NationalInstitute of Allergy and Infectious Diseases at the National Institutesof Health. The manufacturers were full trial collaborators and were apart of the Phase III trial steering committee.

ELISA for Cyclic Peptides and Recombinant gp120 (NK).

This assay was previously described (Haynes et al., “Immune CorrelatesAnalysis of the ALVAC-AIDSVAX HIV-1 Vaccine Efficacy Trial,” N Engl JMed. 366:1275-1286 (2012), which is hereby incorporated by reference inits entirety). Briefly, U-bottom ELISA plates were coated with either 1μg/ml of cyclic peptide (Table 1) or with 3 μg/ml of AIDSVAX recombinantgp120 immunogen A244 or MN.

TABLE 1V2-Related Reagents and Assays Used in the Pilot and Case-Controlled AnalysisUsed for Pilot Case- Assay and Reagents/Lab PI Study controlELISA/Karasavva ^(a)Cyclic V2 (AAs 157-198)

+ + (SEQ ID NO: 66) Cyclic scrambled V2 crown

QVLFKDIHKIVKPLYA

+ + (SEQ ID NO: 69) Cyclic scrambled V2 flanks CENLTDKMFTSR

SESRLDETNYNISC + + (SEQ ID NO: 70) ELISA/Zolla-Pazner^(b)Peptide 1: Most polar V2 LRDKKQRVYSLFYKLDVVQIN +sequence (AAs165-185) (SEQ ID NO: 3) Peptide 2 Most common 40IRDKVQKEYALFYKLDVVPID + AA V2 sequence (SEQ ID NO: 4)Peptide 3 Most polar 40 LRDKKQQVYSLFYRLDIEKIN + AA V2 sequence(SEQ ID NO: 5) Peptide 4 Consensus 40 AA IRDKKQKEYALFYKLDVVPID +V2 sequence (SEQ ID NO: 6) Peptide 6: First 14 AA of LRDKKQRVYSLFYK + +Peptide 1 (AAs 165-178) (SEQ ID NO: 7) Peptide 6G: First 14 AA of GGGLRDKKQRVYSLFYK + Peptide 1 with linker (SEQ ID NO: 10)Peptide 7: Central 14-mer KQRVYSLFYKLDVV + of peptide 1 (SEQ ID NO: 8)Peptide 8: C-term 13-mer YSLFYKLDVVQIN + Peptide 1 (SEQ ID NO: 9)Peptide 17: L165I Mutant GGG I RDKKQRVYSLFYK + of Peptide 6(SEQ ID NO: 11) Peptide 18: K169V Mutant GGG LRDK V QRVYSLFYK +of Peptide 6 (SEQ ID NO: 12) Peptide 19: V172E Mutant GGG LRDKKQR EYSLFYK + of Peptide 6 (SEQ ID NO: 13) Peptide 20: S174A Mutantof Peptide 6 GGG LRDKKQRVY A LFYK + (SEQ ID NO: 14)^(a)Cyclic V2 (AAs 157-198)

(SEQ ID NO: 66) gp70-V1V2 [from subtype

+ + B Case A2]

(SEQ ID NO: 71) ELISA/Berman V2 A244-92TH023 peptide

+ + (SEQ ID NO: 72) V2 MN peptide

+ + (SEQ ID NO: 73) ELISA/Tomaras V2 peptide K178 KK

KKK + (SEQ ID NO: 74) SPR/Rao Cyclic V2 scrambled crown

QVLFKDIHKIVKPLYA

+ + (SEQ ID NO: 69) ^(a)Cyclic V2 (AAs 157-198)

+ + (SEQ ID NO: 66) Luminex/Tomaras IgG binding to biotiny- KK

KKK + + latedV2 peptide K178 (SEQ ID NO: 74) IgA binding to biotiny- KK

KKK + + latedV2 peptide K178 (SEQ ID NO: 74) *Hotspot/Montefiori + +^(a)“Cyclic V2 (amino acids 157-198)” was used in assays in three labsas shown. Throughout this table, bold italicized V2 sequences areidentical to the subtype E 92TH023 used in the prime; underlinedsequences represent scrambled sequences or linkers; italicized sequencesrepresent the sequence the central amino acidss in an extremely polar V2in subtype A strain QB585.2102M.Ev1v5.C with individual mutations shownin bold; plain black represents sequences chosen for particularproperties, as described bold underlined sequences represent the V1V2from subtype B Case A2 (Pinter et al., “Potent Neutralization of PrimaryHIV-1 Isolates By Antibodies Directed Against Epitopes Present in theV1N2 Domain of HIV-1 gp120,” Vaccine 16: 1803-1811(1998), which ishereby incorporated by reference in its entirety) and the central 23-merof V2 from subtype B strain MN. ^(b)All peptides were biotinylated atthe N-terminus with the exception of peptide K178 and peptide V2A244-92TH023 which were biotinylated at the C-terminus. *Multiple V2peptides from various strains (see Table 1 and Karasavvas et al., “TheThai Phase Iii Clinical Trial (RV144) Induces the Generation ofAntibodies that Target a Conserved Region Within the V2 Loop of gp120;The Thai Phase Iii Clinical Trial (RV144) Induces the Generation ofAntibodies that Target a Conserved Region Within the V2 Loop of gp120”,Abstract OA07.08 LB; Bangkok, Thailand pp. OA07.08 LB (2011), which ishereby incorporated by reference in its entirety).

After washing, two-fold serial dilutions of plasma at an initialdilution of 1:100 or, alternatively, anti-V2 human monoclonal antibodies2158 or 697-D (Gorny et al., “Human Anti-V2 Monoclonal Antibody ThatNeutralizes Primary But Not Laboratory Isolates of HIV-1,” J Virol.68:8312-8320 (1994); Pinter at al., “The V1/V2 Domain of gp120 is aGlobal Regulator of Sensitivity of Primary Human Immunodeficiency VirusType 1 Isolates to Neutralization by Antibodies Commonly Induced UponInfection,” J Virol. 78:5205-5215 (2004), each of which is herebyincorporated by reference in its entirety) were used at concentrationsof 0.002-10 μg/ml. Color was developed with HRP-conjugated goatanti-human IgG and substrate, and read A405 nm. The background value wasdetermined from wells that did not contain recombinant proteins orpeptides.

Biotinylated Linear Peptide ELISAs (SZP).

This assay was previously described (Haynes et al., “Immune CorrelatesAnalysis of the ALVAC-AIDSVAX HIV-1 Vaccine Efficacy Trial,” N Engl JMed. 366:1275-1286 (2012), which is hereby incorporated by reference inits entirety). Briefly, StreptaWell ELISA plates (Roche) were coatedwith 1 μg/ml of one of several N-terminus biotinylated linear V2peptides (Table 1); the plates were washed and incubated with RV144plasma specimens diluted 1:100 in RPMI medium containing 15% fetalbovine sera. Alkaline phosphatase (AP)-conjugated goat anti-human IgGand diethanolamine substrate were used to develop color which was readat A405 nm. At each step, every well contained 50 al; specimens were runin duplicate in each experiment, and three experiments were performed.

Binding ELISA with gp70-V1V2 (SZP).

This method was previously described (Haynes et al., “Immune CorrelatesAnalysis of the ALVAC-AIDSVAX HIV-1 Vaccine Efficacy Trial,” N Engl JMed. 366:1275-1286 (2012), which is hereby incorporated by reference inits entirety). Briefly, plates were coated with 1 μg/ml gp70-V1V2 (Table1 and Pinter et al., “Potent Neutralization of Primary HIV-1 Isolates byAntibodies Directed Against Epitopes Present in the V1/V2 Domain ofHIV-1 gp120,” Vaccine 16:1803-1811 (1998), which is hereby incorporatedby reference in its entirety), washed, and then incubated for 1.5 h at37° C. with RV144 plasma diluted 1:100 in RPMI containing 15% fetalbovine sera. After further washing, bound antibodies were visualizedusing AP-conjugated goat anti-human IgG and diethanolamine substrate,and read at 405 nm. At each step, every well contained 50 al; specimenswere run in duplicate in each experiment, and three experiments wereperformed.

V2 Linear Peptide ELISA (PB).

These assays were performed as previously described (Gilbert et al.,“Correlation Between Immunologic Responses to a Recombinant Glycoprotein120 Vaccine and Incidence of HIV-1 Infection in a Phase 3 HIV-1Preventative Vaccine Trial,” J Infect Dis. 191:666-677 (2005), which ishereby incorporated by reference in its entirety). Briefly, plates werecoated with 0.5 μg/well of peptide (Table 1) and incubated overnight at4° C. Three-fold dilutions of test sera were run in duplicate using astarting dilution of 1:30. HRP-labeled anti-human IgG and substrate(OPD) were used to develop color.

ELISAs of Linear and Cyclic Peptides and gp70-V1V2 (GT).

Direct binding ELISAs were conducted as previously described (Haynes etal., “Immune Correlates Analysis of the ALVAC-AIDSVAX HIV-1 VaccineEfficacy Trial,” N Engl J Med. 366:1275-1286 (2012), which is herebyincorporated by reference in its entirety) in 384-well ELISA platescoated with 2 μg/ml of linear or cyclic V2 peptides or gp70-V1V2 andincubated with three-fold serial dilutions of plasma at a startingdilution of 1:50, followed by washing and incubation with 10 μl ofHRP-conjugated goat anti-human Ig secondary antibody and substrate(SureBlue Reserve™). Plates were read at 450 nm.

Overlapping Peptide Microarray Assay (DM). The arrays measuredreactivity with 15-mer peptides with 12 residue overlaps. Raw peptidemicroarray data were processed and analyzed as described in Tomaras etal., “Initial B-cell Responses to Transmitted Human ImmunodeficiencyVirus Type 1: Virion-Binding Immunoglobulin M (IgM) and IgG AntibodiesFollowed by Plasma Anti-gp41 Antibodies With Ineffective Control ofInitial Viremia,” J Virol. 82:12449-12463 (2008), which is herebyincorporated by reference in its entirety. Peptide sequences wereprovided by LANL to cover the entire gp160 HIV Env from six HIV-1 GroupM subtypes (A, B, C, D, CRF01_AE and CRF02_AG) for a total of 1423peptides. The specific peptides were determined by LANL's method forgenerating the mosaic peptide set (Ngo et al., “Identification andTesting of Control Peptides for Antigen Microarrays,” Journal ofImmunological Methods. 343:68-78 (2009), which is hereby incorporated byreference in its entirety) and were manufactured by JPT PeptideTechnologies (Berlin, Germany).

Surface Plasmon Resonance (MR).

Measurements were conducted with a Biacore® T100 as previously described(Haynes et al., “Immune Correlates Analysis of the ALVAC-AIDSVAX HIV-1Vaccine Efficacy Trial,” N Engl J Med. 366:1275-1286 (2012), which ishereby incorporated by reference in its entirety). Briefly, lysozyme(reference surface) and streptavidin (for peptide capture) wereimmobilized onto CM5 chips. Biotinylated V2 peptides (1 μM) (Table 1)were manually injected over the streptavidin-coated chip surface.Heat-inactivated plasma samples diluted 1:50 were injected over the chipsurface followed by a dissociation period, after which a 50 nM solutionof affinity-purified γ-chain-specific sheep anti-human IgG was passedover the peptide coated-Ig bound surface. Non-specific binding wassubtracted and data analysis was performed using BIAevaluation™ 4.1software. Case-control samples were run in triplicate.

IgG and IgA Binding Multiplex Assays (GT). These assays were performedas previously described (Haynes et al., “Immune Correlates Analysis ofthe ALVAC-AIDSVAX HIV-1 Vaccine Efficacy Trial,” N Engl J Med.366:1275-1286 (2012); Tomaras et al., “Initial B-cell Responses toTransmitted Human Immunodeficiency Virus Type 1: Virion-BindingImmunoglobulin M (IgM) and IgG Antibodies Followed by Plasma Anti-gp41Antibodies With Ineffective Control of Initial Viremia,” J Virol.82:12449-12463 (2008), each of which is hereby incorporated by referencein its entirety) using peptide K178 which represents a linear portion ofV2 from immunogens A244 and 92TH023 (Table 1). HIV-specific antibodyisotypes were detected with goat anti-human IgA and mouse anti-humanIgG.

Statistical Analyses.

Immune biomarkers measured two weeks after the last immunizing dose wereassessed as correlates of subsequent infection risk using the previouslydescribed statistical analysis plan (Haynes et al., “Immune CorrelatesAnalysis of the ALVAC-AIDSVAX HIV-1 Vaccine Efficacy Trial,” N Engl JMed. 366:1275-1286 (2012), which is hereby incorporated by reference inits entirety). Briefly, for each immune biomarker, logistic regressionaccounting for the sampling design was used to estimate the odds ratio(OR) of infection, controlling for gender and baseline behavioral risk.The OR was estimated both for the immune biomarker as a categoricalvariable and for variables with greater than 50% of vaccinees exhibitinga positive response, as a quantitative variable (scaled to have a SD=1).For the categorical analysis, if the positive response rate is less than50%, then the OR compares positive vs. negative responders. If thepositive response rate is 50-85%, then the OR compares high vs. negativewhere high is above the median of the positive responders. For positiveresponse rates>85%, the OR compares high vs. low where high and low arethe upper and bottom tertiles of the response for vaccine recipients.The statistical analysis plan was finalized before data analysis and isdescribed in detail in Haynes et al., “Immune Correlates Analysis of theALVAC-AIDSVAX HIV-1 Vaccine Efficacy Trial,” N Engl J Med. 366:1275-1286(2012), which is hereby incorporated by reference in its entirety.

The lasso model selection procedure (Friedman et al., “RegularizationPaths for Generalized Linear Models Via Coordinate Descent,” J StatSoftw. 33:1-22 (2010), which is hereby incorporated by reference in itsentirety) as implemented in the R software package was used to assessthe ability of 12 V2 variables from Table 2 to predict infection whenincluded in a multivariate logistic model adjusting for gender andbehavioral risk score.

TABLE 2 Response Rate and Odds Ratios (ORs) Calculated From the CaseControl Specimens Tested With 13 V2 Variables. Quantitative CategoricalInves- P P Assay tigator Institution OR^(1,2) value OR^(1,3) value V2cyclic Nicos AFRIMS peptides-ELISA Karasavvas Cyclic V2 NA NA 0.82 0.63(AAs 157-198) Cyclic V2 NA NA NA NA scrambled crown Cyclic V2 0.76 0.100.84 0.66 scrambled flanks V2 cyclic Mangala USMHRP peptides-SPR RaoCyclic V2 0.79 0.18 0.90 0.80 scrambled crown Cyclic V2 0.81 0.24 0.840.66 (AAs 157-198) V2 reagents- Susan New York ELISA Zolla- UniversityPazner Cyclic V2 0.82 0.26 0.65 0.31 (AAs 157-198) Biotin V2 Peptide0.95 0.80 0.85 0.76 6 (AAs 165-178) gp70-V1V2 0.70 0.06 0.43 0.06 V2linear Philip University peptides-ELISA Berman of California, Santa CruzV2 MN peptide NA NA 0.41 0.25 V2 A244-92TH023 0.90 0.57 0.88 0.74peptide IgA and IgG Abs Georgia Duke vs. V2 peptide- Tomaras UniversityLuminex IgA V2 A244 NA NA 0.79 0.77 K178 peptide IgG V2 A244 NA NA 1.020.95 K178 peptide Peptide David Duke Microarray Montefiori University V2Hotspot 0.64 0.03 0.32 0.02 analysis ¹Estimated odds ratios are computedusing a logistic regression model accounting for the sampling design andadjusting for gender and behavioral risk score, as described in Hayneset al., “Immune Correlates Analysis of the ALVAC-AIDSVAX HIV-1 VaccineEfficacy Trial,” N Engl J Med. 366:1275-1286 (2012), which is herebyincorporated by reference in its entirety. ²Estimated odds ratio per onestandard deviation increment in the immune biomarker; not available (NA)if response rates, when applicable, are less than 50%. For example, theOR of 0.70 (ELISA binding to gp7O-V1V2) means that for every higher SDvalue, the rate of infection is reduced by 30%, while the OR of 0.43means that vaccinees with responses in the upper third had an infectionrate 57% lower than vaccinees with responses in the lower third.³Estimated odds ratios comparing subgroups defined by high vs. lowresponses except for two (IgA V2 A244 K178 and V2 MN) which comparepositive vs. negative response and one (biotin V2 peptide 6) whichcompares high vs. negative; not available (NA) for Cyclic V2 scrambledcrown (ELISA) which has no positive responses.

The cyclic V2 scrambled crown variable was excluded, because it had nopositive responses. Two of the variables with low response rates, IgA V2A244 K178 peptide and V2 MN peptide, were dichotomized as 1 for responseand 0 for non-response, while the remaining ten variables were includedon a quantitative scale. The best parsimonious model was chosen based onthe average area under the receiver operating characteristic curvederived from 1,000 10-fold cross-validation splits.

Example 1—Antigenicity of the Boosting Immunogens Used

In RV144, the V2 sequence in the recombinant ALVAC priming immunogenderived from subtype E strain 92TH023 was:

(SEQ ID NO: 66) ¹⁵⁷CSFNMTTELRDKKQKVHALFYKLDIVPIEDNTSS.SEYRLINC¹⁹⁸The V2 sequence in the protein boosting gp120 immunogen AIDSVAX E(strain A244) was:

(SEQ ID NO: 67) ¹⁵⁷CSFNMTTELRDKKQKVHALFYKLDIVPIEDNNDS.SEYRLINC¹⁹⁸The V2 sequence of the protein boosting gp120 immunogen AIDVAX B (strainMN) was:

(SEQ ID NO: 68) ¹⁵⁷CSFNITTSIGDKMQKEYALLYKLDIEPI.DN.DSTS.YRLISC¹⁹⁸Insertion of periods in the sequences allows for alignment. Numberingshown and used throughout this report is that assigned to strain HxB2(Ratner et al., “Complete Nucleotide Sequences of Functional Clones ofthe AIDS Virus,” AIDS Res Hum Retroviruses 3:57-69 (1987), which ishereby incorporated by reference in its entirety).

The antigenic reactivity of the V2 region in AIDSVAX B and E wasassessed using human anti-V2 monoclonal antibodies 697D and 2158 (Gornyet al., “Human Anti-V2 Monoclonal Antibody That Neutralizes Primary ButNot Laboratory Isolates of HIV-1,” J Virol. 68:8312-8320 (1994); Pinterat al., “The V1/V2 Domain of gp120 is a Global Regulator of Sensitivityof Primary Human Immunodeficiency Virus Type 1 Isolates toNeutralization by Antibodies Commonly Induced Upon Infection,” J Virol.78:5205-5215 (2004), each of which is hereby incorporated by referencein its entirety). As shown in FIG. 1, the titration curves for each ofthese monoclonal antibodies with the two boosting immunogens could besuperimposed, with half-maximal binding achieved at 0.0057 and 0.0055μg/ml of monoclonal antibody 697D, and 0.0041 and 0.0039 μg/ml ofmonoclonal antibody 2158 vs. AIDSVAX A244 and AIDSVAX MN, respectively.This analysis suggests that, with respect to the highly conformationalV2 epitopes recognized by these monoclonal antibodies (Gorny et al.,“Functional and Immunochemical Cross-Reactivity of V2-specificMonoclonal Antibodies from Human Immunodeficiency Virus Type 1-infectedIndividuals,” Virology 427: 198-207 (2012); Pinter et al., “The V1/V2Domain of gp120 is a Global Regulator of Sensitivity of Primary HumanImmunodeficiency Virus Type 1 Isolates to Neutralization by AntibodiesCommonly Induced Upon Infection,” J Virol. 78: 5205-5215 (2004); Gorny,“Production of Human Monoclonal Antibodies Via Fusion of Epstein-BarrVirus-Transformed Lymphocytes with Heteromyeloma,” In: Celis, editor.In: Cell Biology: A Laboratory Handbook: Academic Press 276-281 (1994),each of which is hereby incorporated by reference in its entirety), theantigenicity of the A244 and MN gp120 immunogens are similar. Notably,these two monoclonal antibodies also bind to gp70-V1V2 (Gorny et al.,“Functional and Immunochemical Cross-Reactivity of V2-specificMonoclonal Antibodies from Human Immunodeficiency Virus Type 1-infectedIndividuals,” Virology 427: 198-207 (2012); Karasavvas et al., “The ThaiPhase Iii Clinical Trial (RV144) Induces the Generation of Antibodiesthat Target a Conserved Region Within the V2 Loop of gp120; The ThaiPhase Iii Clinical Trial (RV144) Induces the Generation of Antibodiesthat Target a Conserved Region Within the V2 Loop of gp120,” AbstractOA07.08 LB; Bangkok, Thailand. pp. OA07.08 LB (2011), each of which ishereby incorporated by reference in its entirety).

Example 2—The V2 Antibody Response in RV144 can be Detected with BothLinear and V1V2-Scaffolded Antigens

A gp70-V1V2 scaffolded protein carrying the V1 and V2 loops from a cladeB strain, case A2, was previously described (Pinter et al., “PotentNeutralization of Primary HIV-1 Isolates by Antibodies Directed AgainstEpitopes Present in the V1/V2 Domain of HIV-1 gp120,” Vaccine16:1803-1811 (1998), which is hereby incorporated by reference in itsentirety). When Set C plasma specimens (from 20 placebo and 80 vaccinerecipients) were tested in the pilot studies at a dilution of 1:100,none of the specimens from the placebo recipients contained detectableantibodies to gp70-V1V2. In contrast, the plasma of 67 of 80 (84%)vaccine recipients contained antibodies reactive with this reagent (FIG.2). Moreover, the dynamic range of the assay was large, covering anoptical density range from the cut-off, 0.276 OD units, to 1.918. Arelatively poor correlation was found between this assay and otherassays that measured various V2 variables, suggesting that the antibodyresponse measured with gp70-V1V2 represents a unique “immunologic space”(FIG. 3).

The frequency of V2 responses detected with pilot study specimensderived from vaccinees varied with the assay used, ranging from 6% forIgA antibodies reactive with a linear V2 peptide (K178) when measured byLuminex (see Table 1 and Haynes et al., “Immune Correlates Analysis ofthe ALVAC-AIDSVAX HIV-1 Vaccine Efficacy Trial,” N Engl J Med.366:1275-1286 (2012), which is hereby incorporated by reference in itsentirety) to 97% for IgG antibodies reactive with an A244 (subtype E)cyclic V2 peptide (see Table 1 and Karasavvas et al., “The Thai PhaseIii Clinical Trial (RV144) Induces the Generation of Antibodies thatTarget a Conserved Region Within the V2 Loop of gp120; The Thai PhaseIii Clinical Trial (RV144) Induces the Generation of Antibodies thatTarget a Conserved Region Within the V2 Loop of gp120”, Abstract OA07.08LB; Bangkok, Thailand. pp. OA07.08 LB (2011), which is herebyincorporated by reference in its entirety).

When reactivity to various V2 reagents were compared in parallel byELISA, the response to the K178 peptide was significantly stronger thanthat to gp70-V1V2 or to cyclic V2 peptide (amino acids 157-198), asshown in FIG. 4. Thus, the RV144 vaccine induced antibodies that reactedto both scaffolded-V1V2 and to linear V2 peptides, but the response tothe latter appears to be stronger.

Example 3—Delineation of the Linear V2 Epitopes Recognized by PlasmaAntibodies from Vaccinees

For fine mapping of linear V2 epitopes recognized by antibodies in theplasma of RV144 vaccinees, four 21-mer peptides (Peptides 1-4 (SEQ IDNOS: 3 to 6) in Table 1 and FIG. 5A) were selected on the basis of abioinformatics analysis of V2 sequences from the LANL HIV Database.Peptide 1 (SEQ ID NO: 3) was derived from the V2 of a strain with thehighest number of polar amino acids (subtype A strainQB585.2102M.Ev1v5.C from Kenya); this V2 was 38 amino acids in length.Since V2 is most frequently 40 amino acids in length (Zolla-Pazner etal., “Structure-Function Relationships of HIV-1 EnvelopeSequence-Variable Regions Provide a Paradigm for Vaccine Design. Nat RevImmunol. 10:527-535 (2010), which is hereby incorporated by reference inits entirety), further analyses identified sequences from virusescontaining V2 regions 40 amino acids long: Peptide 2 (SEQ ID NO: 4)represents the central 21 amino acids of V2 in the most common naturallyoccurring sequence (derived from subtype B strain 878v3_2475). Peptide 3(SEQ ID NO: 5) is the V2 with the highest number of polar amino acids(from subtype A strain 01TZA341). Peptide 4 (SEQ ID NO: 6) is theconsensus V2 sequence among all viruses with V2 regions of 40 aminoacids.

As illustrated by the ELISA data (FIG. 5A), Peptide 1 (SEQ ID NO: 3) wasthe most reactive with plasma from the RV144 vaccinees: 61% of plasmashowed positive reactivity, i.e., had OD values above the cut-off whichwas based on the mean+3 SD of control plasma (from placebo recipients).Peptide 3 (SEQ ID NO: 5) was also reactive (49% positive), whilePeptides 2 (SEQ ID NO: 4) and 4 (SEQ ID NO: 6) were poorly reactive (0%and 7% positive, respectively). Three overlapping peptides (Peptides 6-8(SEQ ID NOS: 7 to 9)) were synthesized based on the sequence of the moststrongly reactive Peptide 1 (SEQ ID NO: 3). Results with the overlappingpeptides showed that the epitope maps to the 14 residues in Peptide 6(SEQ ID NO: 7) containing amino acids 165-178 (FIG. 5A). The residues inthis V2 region form the outer C strand of the 3-sheet folded domain ofVV2 (FIG. 6) (McLellan et al., “Structure of HIV-1 gp120 V1/V2 DomainWith Broadly Neutralizing Antibody PG9,” Nature 480:336-343 (2011),which is hereby incorporated by reference in its entirety).

To distinguish the amino acids that play a critical role in determininganti-V2 antibody reactivity, a further series of peptides was designed.Results with these peptides indicated that L165I (Peptide 17 (SEQ ID NO:11)) and S174A (Peptide 20 (SEQ ID NO: 14)) replacements had littleeffect on reactivity (FIG. 5B). In contrast, the K169V (Peptide 18 (SEQID NO: 12)) and the V172E (Peptide 19 (SEQ ID NO: 13)) replacementsprofoundly reduced the reactivity of the plasma, indicating that thesetwo residues are critical for binding of V2 peptides by thevaccine-induced antibodies. These data acquire enhanced importance inlight of results showing that (a) sieve analysis showed that a residueother than K at position 169 was more frequent in breakthrough virusesin vaccine recipients (Rolland et al., “Sequence Analysis of HIV-1Breakthrough Infections in the RV144 Trial. Characterization ofBreakthrough Viruses,” Sequence Analysis of HIV-1 BreakthroughInfections in the RV144 Trial, Abstract S07.02 S07.02 (2011) andEdlefsen et al., “Sieve Analysis of RV144,” Abstract S07.04 S07.04(2011), each of which is hereby incorporated by reference in itsentirety), as well as (b) Gln (E) strongly predominates in subtype Ewhereas valine (V) slightly predominates at position 172 in subtype B.Notably, the poor response to Peptide 19 (SEQ ID NO: 13), in which theV172E mutation appears, suggests that the V2 antibody response wasinduced by the subtype E (A244) gp120 boosting immunogen rather than thesubtype B (MN) immunogen.

Example 4—Comparison of V2 Assays Run in the Case-Control Study

Based on the pilot studies, six assay types were chosen for measuring 13variables with case-control specimens (Table 2). One of these (ELISAbinding to gp70-V1V2) was chosen as a primary variable. The secondaryvariables provided by the 12 additional V2 assays were run inexploratory analyses with case-control specimens. The primary andsecondary variables were chosen to represent assays whose results didnot correlate with one another. The heat map in FIG. 3 represents theSpearman rank correlations between the V2 assays, and demonstrates thatthe primary variable, binding of IgG antibodies to gp70-V1V2, correlatedonly weakly with ELISA binding to cyclic V2 (amino acids 157-198)(Spearman correlation: 0.5-0.6). Analysis of the microarray “hotspot”data was not performed until after completion of the analysis of theinitial pilot and case-control studies. Interestingly, the V2 hotspotvariable correlates poorly with all of the other V2 variables.

For the univariate analysis, as in the multivariate analysis (Haynes etal., “Immune Correlates Analysis of the ALVAC-AIDSVAX HIV-1 VaccineEfficacy Trial,” N Engl J Med. 366:1275-1286 (2012), which is herebyincorporated by reference in its entirety), statistical significance wasapproached or achieved with the primary variable of ELISA binding togp70-V1V2 (p=0.06 and p=0.02, respectively). With one of the secondaryvariables (V2 hotspot), significance was achieved (p=0.03 quantitative,and p=0.02 categorical, Table 2). The ORs calculated for each V2variable are shown in FIG. 7 and Table 2. The univariate ORs for all V2variables were <1.02, compatible with the hypothesis that V2 antibodiesplayed a role in reducing infection. When binding antibodies wereassessed by peptide microarray analysis using linear overlappingpeptides covering the entire V2 region of seven major genetic subtypes,the lowest and most statistically significant OR was achieved (FIG. 7and Table 2). The results are shown in FIG. 8 for V2 residues 160-183.Most of the remaining C-terminal portion of V2 is poorly immunogenic(Zolla-Pazner et al., “Structure-Function Relationships of HIV-1Envelope Sequence-Variable Regions Provide a Paradigm for VaccineDesign,” Nat Rev Immunol. 10:527-535 (2010), which is herebyincorporated by reference in its entirety), and similarly, there waslittle or no reactivity with peptides in the V1 region. The aggregateresponse (FIG. 8A), shows that the V2 response is centered aroundresidue K¹⁷³. The peptides with the strongest reactivity encompassresidues 163-177 (FIG. 8B), which matches the results from theindependent ELISA data described above. It is also noteworthy that theweakest reactivity in the microarray analysis was detected with thesubtype B subset of V2 peptides, confirming the poor reactivity withPeptide 19 (SEQ ID NO: 13) (FIG. 5B) which represents a canonicalsubtype B V2 containing a Glu (E) at position 172. Interestingly, thereactivity with the V2 peptide representing the sequence of vaccinestrain MN (subtype B) also includes E¹⁷². Only 24 of 246 vaccinees'specimens reacted with the V2 MN peptide, again confirming the poorreactivity with the subtype B V2; however, strikingly, none of these 24vaccinees were infected, resulting in an OR for positive vs. negativeresponders of 0.41 (Table 2 and FIG. 7). The 0.25 p-value reflects thelow power of these data due to the very few positive responders andcould also be due to poor sensitivity of the assay since the resultswere reported as endpoint titers after a starting dilution of 1:30.

Discussion of Examples 1-4

In this study, the results achieved with the entire panel of V2 assaysused in the RV144 pilot and case-control studies were probed in order tounderstand more fully the nature of the V2 antibody response and why thehigh response to epitopes in this region is associated with a lower rateof infection in vaccinees. The data presented include all of the datadescribing the V2 antibodies induced by the vaccine and available fromboth the pilot and the case-control specimens.

These studies document at least two types of V2 antibodies induced bythe RV144 vaccine: antibodies reactive with a scaffolded V1V2 protein,gp70-V1V2, and antibodies specific for linear V2 peptides. Studies withhuman monoclonal antibodies suggest that these may be non-overlappingantibody populations since monoclonal antibodies such as 697D and 2158react with conformational V1V2 epitope(s) carried by gp70-V1V2 but notwith linear peptides (Gorny et al., “Functional and ImmunochemicalCross-Reactivity of V2-Specific Monoclonal Antibodies From HumanImmunodeficiency Virus Type 1-Infected Individuals,” Virology427:198-207 (2012); Gorny et al., “Human Anti-V2 Monoclonal AntibodyThat Neutralizes Primary but Not Laboratory Isolates of HIV-1,” J Virol.68:8312-8320 (1994), each of which is hereby incorporated by referencein its entirety), while monoclonal antibodies such as CH58 and CH59react with linear V2 peptides but not with gp70-V1V2. The primaryvariable that correlated with reduced risk of infection measuredantibody activity in ELISA with gp70-V1V2. This reagent retains aconformation presented in vivo during infection since it is recognizedby antibodies in sera of infected individuals and was used for theselection of two monoclonal antibody-producing hybridomas from the cellsof HIV-infected individuals which are broadly cross-reactive withdiverse envelopes and neutralize several Tier 1 pseudoviruses (Gorny etal., “Functional and Immunochemical Cross-Reactivity of V2-SpecificMonoclonal Antibodies From Human Immunodeficiency Virus Type 1-InfectedIndividuals,” Virology 427:198-207 (2012); Pinter et al., “The V1/V2Domain of gp120 is a Global Regulator of Sensitivity of Primary HumanImmunodeficiency Virus Type I Isolates to Neutralization by AntibodiesCommonly Induced Upon Infection,” J Virol. 78:5205-5215 (2004), each ofwhich is hereby incorporated by reference in its entirety).

The reactivity of vaccinees' antibodies with overlapping V2 peptidesalso correlated with reduced risk of infection (Table 2), generating thehypothesis that antibodies to linear V2 epitopes were also involved inreducing the rate of HIV infection. The observation that none of thevaccinees who produced antibodies reactive with the linear subtype B MNV2 peptide were infected with HIV during the trial is intriguing,although the low power of the result reduces confidence in thesignificance of this observation. The data generated with various linearV2 peptides indicate that: (a) the dominant immunogenic linear V2epitope in the RV144 vaccine encompasses residues 165 to 178; (b) the V2antibodies were induced primarily by subtype E A244 rather than thesubtype B MN gp120 boost; (c) the V2 antibodies were cross-reactive withV2 peptides derived from several subtypes, (d) the dominant linear V2epitope was located in the C β-strand of the V1V2 complex (FIG. 6 andMcLellan et al., “Structure of HIV-1 gp120 V1/V2 Domain With BroadlyNeutralizing Antibody PG9” Nature 480: 336-343 (2011), which is herebyincorporated by reference in its entirety), (e) residues K¹⁶⁹ and V¹⁷²,were critical for the binding of vaccinees' plasma antibodies to V2peptides, and (f) the V2 epitope includes the lysine at position 169which was identified by sieve analysis to be mismatched in breakthroughinfections (Rolland et al., “Sequence Analysis of HIV-1 BreakthroughInfections in the RV144 Trial. Characterization of BreakthroughViruses,” Sequence Analysis of HIV-1 Breakthrough Infections in theRV144 Trial, Abstract S07.02 S07.02 (2011), which is hereby incorporatedby reference in its entirety).

It is noteworthy that the single primary variable showing an inversecorrelate of infection risk in the RV144 case-control study was antibodyreactivity with gp70-V1V2 which contains the V1V2 sequence of case A2, asubtype B strain (Pinter et al., “Potent Neutralization of Primary HIV-1Isolates by Antibodies Directed Against Epitopes Present in the V1/V2Domain of HIV-1 gp120,” Vaccine 16:1803-1811 (1998), which is herebyincorporated by reference in its entirety) which carries both the V169and the E172 residues that reduce reactivity of vaccinees' antibodieswith V2 peptides. Notably, however, as shown above, vaccinees' plasma doreact with subtype B-derived linear V2 peptides, though with less potentand less frequent reactivity than with V2 peptides from other subtypes.The data with gp70-V1V2 and the linear peptides may suggest that theeffective antibody populations are those which are broadlycross-reactive, targeting conformational and linear epitopes shared bydiverse HIV-1 subtypes. Indeed, these data, together with bioinformaticsdata on V2 (Zolla-Pazner et al., “Structure-Function Relationships ofHIV-1 Envelope Sequence-Variable Regions Provide a Paradigm for VaccineDesign,” Nat Rev Immunol. 10:527-535 (2010), which is herebyincorporated by reference in its entirety) and studies of thepreferential gene usage of VH families by V2-specific monoclonalantibodies (Gorny et al., “Functional and ImmunochemicalCross-Reactivity of V2-Specific Monoclonal Antibodies From HumanImmunodeficiency Virus Type 1-Infected Individuals,” Virology427:198-207 (2012), which is hereby incorporated by reference in itsentirety), support the presence of conserved and immunologicallycross-reactive elements in the V2 loop. The role of shared structuresand antigenic determinants in the variable loops of the envelope ininducing potentially protective antibody responses is also suggested bythe involvement of the V2 and V3 loops as components of the epitopesrecognized by the class of potently neutralizing antibodies that targetquaternary epitopes and proteoglycans on the envelope spike (Gorny etal., “Identification of a New Quaternary Neutralizing Epitope on HumanImmunodeficiency Virus Type I Virus Particles,” J Virol. 79:5232-5237(2005); Walker et al., “Broad and Potent Neutralizing Antibodies From anAfrican Donor Reveal a New HIV-1 Vaccine Target,” Science 326:285-289(2009); Changela et al., “Crystal Structure of Human Antibody 2909Reveals Conserved Features of Quaternary-Specific Antibodies thatPotentially Neutralize HIV-1,” J Virol. 85:2524-2535 (2011); Spurrier etal., “Structural Analysis and Computational Modeling of Human andMacaque Monoclonal Antibodies Provide a Model for the QuaternaryNeutralizing Epitope of HIV-1 gp120,” Structure 19:691-699 (2011); Wu etal., “Immunotypes of a Quaternary Structure of the HIV-1 Envelope AffectViral Vulnerability to Neutralizing Antibodies,” J Virol. 85:4578-4585(2011); Bonsignori et al., “Analysis of a Clonal Lineage of HIV-1Envelope V2/V3 Comformational Epitope-Specific Broadly NeutralizingAntibodies and Their Inferred Unmutated Common Ancestors,” Journal ofVirology 85:9998-10009 (2011); Walker at al., “Broad NeutralizationCoverage of HIV by Multiple Highly Potent Antibodies,” Nature477:466-470 (2011); Pejchal et al., “A Potent and Broad NeutralizingAntibody Recognizes and Penetrates the HIV Glycan Shield,” Science334:1097-1103 (2011), each of which is hereby incorporated by referencein its entirety).

The explanation for the strong induction of V2 antibodies by the A244subtype E gp120 immunogen compared to the weak response induced by theMN subtype B gp120 despite the similar antigenicity of the two proteinshas several possible explanations. It may be due to a proteolyticcleavage site in the V2 loop of MN; a cathepsin D cleavage site (QKEYALL(SEQ ID NO: 75)) exists in the V2 of MN (Yu et al., “Protease CleavageSites in HIV-1 gp120 Recognized by Antigen Processing Enzymes areConserved and Located at Receptor Binding Sites,” J Virol. 84:1513-1526(2010), which is hereby incorporated by reference in its entirety),while this site is absent from the V2 of A244 (QKVHALF (SEQ ID NO: 76)).The importance of proteoloysis by lysosomal enzymes on antigenpresentation and induction of immune responses to gp120 was previouslydescribed (Chien et al., “Human Immunodeficiency Virus Type I EvadesT-helper Responses by Expoiting Antibodies that Suppress AntigenProcessing,” J Virol. 78:7645-7652 (2004), which is hereby incorporatedby reference in its entirety), providing a theoretical explanation forunderstanding the differential antibody responses to these two gp120immunogens. Alternatively, the immunogenicity of the V2 in the MN gp120boosting immunogen may be less than that of the A244 immunogen, and/orthe subtype E rather than the subtype B V2 region is the greatersimilarity of the AIDSVAX subtype E gp120 protein boost to the subtype EEnv used in the prime. To address these issues, further studies of V2responses with specimens from other vaccine trials, e.g., VAX003 andVAX004, are underway, along with assays using additional peptides,proteoglycans, and epitope-scaffolded proteins.

Finally, the mechanisms by which anti-V2 antibodies may reduce HIVinfection have yet to be understood. As noted, anti-V2 monoclonalantibodies can neutralize many Tier 1 pseudoviruses in the TZM.bl assay(Gorny et al., “Functional and Immunochemical Cross-Reactivity ofV2-Specific Monoclonal Antibodies From Human Immunodeficiency Virus Type1-Infected Individuals,” Virology 427:198-207 (2012), which is herebyincorporated by reference in its entirety). It is possible that theymediate broader neutralizing activity than is detected in thisparticular assay. Plasma samples from RV144 neutralized some Tier 1viruses in the TZM-bl assay and in a more sensitive assay with A3R5cells; however no neutralization of Tier 2 viruses was detected ineither assay. Since V2 can be detected on the surface of virions (Nyambiet al., “Conserved and Exposed Epitopes on Intact, Native, Primary HumanImmunodeficiency Virus Type I Virions of Group M,” J Virol. 74:7096-7107(2000), which is hereby incorporated by reference in its entirety) andinfected cells (Zolla-Pazner et al., “Serotyping of Primary HumanImmunodeficiency Virus Type I Isolates From Diverse Geographic Locationsby Flow Cytometry,” J Virol. 69:3807-3815 (1995), which is herebyincorporated by reference in its entirety), these antibodies may alsomediate various other anti-viral functions such as ADCC, ADCVI,virolysis, virus opsonization, virus aggregation, etc. Along withcurrent studies of the potential biologic functions of V2 antibodies,assessment is on-going to test several hypotheses, including those thatpostulate that anti-V2 antibodies prevent conformational changes in theenvelope necessary for binding to CCR5, and that these antibodies may,or may not, prevent binding of the envelope to 4037. Interestingly,after vaccination of non-human primates with Ad26 and MVA containingSIVsm543 inserts, a low dose intra-rectal heterologous SIVmac251challenge identified a potential V2 correlate of protection (Barouch etal., “Vaccine Protection Against Acquisition of Neutralization-ResistantSIV Challenges in Rhesus Monkeys,” Nature 482:89-93 (2012), which ishereby incorporated by reference in its entirety). While the relevanceof the SIV model to ALVAC-HIV and AIDSVAX B/E responses in humans may beunclear, the presence of this analogous protective response aftervaccination, in addition to the results of the RV144 immune correlatesanalysis, may provide a means to illuminate the postulated mechanism forreducing the risk of infection.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

1.-39. (canceled)
 40. An isolated antibody raised against an isolatedimmunogenic peptide comprising a V2 loop fragment from HIV surfaceenvelope glycoprotein gp120 which binds specifically with antibodies inblood of patients vaccinated with a vaccine that has shown protectionfrom HIV-1 infection, does not react with blood of matched patients whodid not receive the vaccine, and can, therefore, elicit anti-HIV-1antibodies which protect against HIV-1 infection.
 41. An isolatedantibody raised against an isolated immunogenic peptide comprising a V2loop fragment from HIV surface envelope glycoprotein gp120 which bindsspecifically with antibodies in blood of patients vaccinated with avaccine that has shown protection from HIV-1 infection, does not reactwith blood of matched patients who did not receive the vaccine, and can,therefore, elicit anti-HIV-1 antibodies which protect against HIV-1infection and an immunogenic scaffold protein, wherein said peptide isinserted into said scaffold protein, wherein said polypeptide has aconformation that is recognized by, and bound by, a broadly neutralizinganti-HIV-1 antibody.
 42. An isolated antibody raised against a cyclizedform of an isolated immunogenic peptide comprising a V2 loop fragmentfrom HIV surface envelope glycoprotein gp120 which binds specificallywith antibodies in blood of patients vaccinated with a vaccine that hasshown protection from HIV-1 infection, does not react with blood ofmatched patients who did not receive the vaccine, and can, therefore,elicit anti-HIV-1 antibodies which protect against HIV-1 infection. 43.(canceled)